Abstract. Approximately 2.5 million Americans have rheumatoid arthritis (RA), which is one of the most common chronic autoimmune disorders in the older population. There is no cure for RA, and up to 40% of patients do not respond to current therapies. As a result, disease progression leads to joint destruction and severe disability in the elderly. Therefore, the identification of a novel therapeutic strategy used alone or in combination with existing therapies would resolve a critical barrier in RA treatment. The overall goal of this project is to address this unmet medical need by developing novel small molecule drugs for RA therapy. Our strategy is to build upon two new discoveries: (a) the important role of the toll-like receptor 5 (TLR5) in RA, and (b) a novel chemical series of small molecule TLR5 inhibitors. Preliminary studies revealed that TLR5 is highly elevated in RA myeloid cells as compared to normal myeloid cells, and its expression closely correlates with RA disease activity score (DAS28). Furthermore, ligation of TLR5 to its natural ligand expressed in the joints transforms RA peripheral blood (PB) myeloid cells into proinflammatory M1 macrophages and mature osteoclasts. In mice, systemic and local injection of a TLR5 agonist exacerbates joint swelling and bone erosion. Conversely, anti- TLR5 antibody (Ab) therapy attenuates collagen induced arthritis (CIA) joint inflammation and bone destruction. Coincident with these studies, collaborators discovered small molecule TLR5 inhibitors that antagonize the interaction between TLR5 and its agonist, flagellin, from bacterial infectious agents including Pseudomonas and Burkholderia. Several validated hits from the screen are effective in RA PB myeloid cells. In particular, a 2-iminobenzimidazolene (IBZ) series of compounds abrogates RA M1 macrophage differentiation and osteoclast maturation more effectively than does anti-TNF Ab, anti-IL-6R Ab or a TLR4 antagonist, and several analogs reduced flagellin induced TNF, IL-6 and CCL2 production by up to 70% in RA PB myeloid cells with low cytotoxicity, yielding a selectivity index of >10 and responsive structure?activity relationships (SAR) that will guide further chemical optimization. All three active analogs displayed good stability (90%, 84%, and 74%) in the presence of mouse liver microsomes + NADPH, and the aqueous solubility of all 3 analogs was in excess of 400 M. In Phase I of this translational drug development project, the IBZ series will be chemically optimized to maximize potency and selectivity as well as drug-like absorption, distribution, metabolism and excretion (ADME) properties, and the mechanism of action together with the target specificity will be determined for the IBZ series. Other compounds from the TLR5 inhibition screen at MBX will serve as backup scaffolds for this project. In Phase II, in vivo-validated leads and a preclinical candidate will be identified from the IBZ series by formulating and evaluating prioritized analogs in mice to determine the maximum tolerated dose, pharmacokinetics and efficacy in a murine collagen-induced arthritis model. In Phase III, the effectiveness of the preclinical candidate will be evaluated in comparison with other currently available therapies using acute and chronic RA preclinical models, and Investigational New Drug (IND) enabling toxicology and safety pharmacology studies will be carried out.